PB1975: ABL1 GENE DNA SEQUENCING IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA AND PH+ ACUTE LYMPHOBLASTIC LEUKEMIA.
Authors: Elena Kuzmina, Bella Biderman, Elena Stepanova, Ekaterina Chelysheva, Oleg Shukhov, Anna Petrova, Irina Nemchenko, Margarita Gurianova, Anastasia Bykova, Alina Kohno, Anna Turkina, Andrey Sudarikov
Journal: HemaSphere (2023)

Topic: 8. Chronic myeloid leukemia - Clinical Background: Next generation sequencing (NGS) is widely used for BCR::ABL1 mutations detection in resistant chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) patients (pts). Usually it includes the RNA-based nested PCR and is able to detect low VAF mutations with sensitivity of about 3%, while Sanger Sequencing (SS) sensitivity is about 15-20%. However, RNA is sensitive to denaturation and requires several retro-transcription and amplification steps, which can cause polymerase errors and false-positive results. Meanwhile, DNA-based NGS is amplifying ABL1 from non-rearranged ABL1 allele of tumor cells and ABL1 gene of healthy cells. As DNA is more stable and might better represent the clonal level and evolution, a DNA-based NGS methodology for kinase domain (KD) ABL1 mutation detection and monitoring can be relevant in clinical practice. Recent reports have showed comparable results in mutation detection by both methods. (Sánchez, R, 2022). Aims: To study the DNA-based NGS methodology for KD ABL1 mutation detection in resistant/relapsed CML/Ph+ ALL pts. Methods: In total 39 peripheral blood samples of 30 CML/Ph+ALL pts were collected, 9 pts had two samples at different time points. Most of the samples were from CML pts (37/39). Median of CML pts therapy was 7 years (0,6-21,8), all pts received ≥ 2 tyrosine kinase inhibitors (TKIs) lines and had failure to TKIs treatment. Two pts’ with Ph+ALL were in a relapse at the moment of analysis. DNA was isolated using standard “salting out” procedure. Seven pairs of primers were synthesized for the following ABL1 gene exons: 4, 5, 5 + 6, 6, 7, 8, 9. The resulting amplicons were used to create NGS libraries with Nextera XT kits (Illumina, USA) and were analyzed on the MiSeq genetic analyzer (Illumina, USA). Data filtering, deletion of service sequences, mapping of readings, calling for variants and annotation were carried out using Trimmomatic, BWA, SAMtools, Vardict and Annovar open-source utilities. Variants marked like pathogenic by Franklin by Genoox database were analyzed. Results: The median of BCR::ABL1 IS level of 39 samples was 34% (range 0,1-129%). BCR::ABL1 mutations were detected by DNA-based NGS in 19 (49%) samples: in 13 of them – T315I, other mutation variants were: F359V, Q252H, T315I+E255K, M244V, A337T. The lowest BCR::ABL1 IS level when mutation was detected was 1%. In 37 of 39 samples mutational status simultaneously was evaluated by SS or RNA-based NGS. In summary, 34/39 samples confirmed the same mutational status by DNA-based NGS. Fifteen mutations, detected by DNA-based NGS, matched to SS-control and 3 - to RNA-based NGS-control. One sample with A337T ABL1 mutation in the myristoyl pocket detected by DNA-based NGS at 3% VAF, unfortunately had no control to compare. In 3 samples mutations were not detected by DNA-based NGS, although they were detected by SS/ RNA-based NGS and in serial samples. Clonal dynamic in pts with paired samples is presented in a table 1. Summary/Conclusion: A technology of DNA-based NGS alternative to standard RNA-based approaches has shown promising results in KD ABL1 mutations detection, with results highly matching to SS or RNA-based NGS detection. The method is reproducible and can be easily implemented in the laboratory routines. The advantages of the technology are evident in the conditions of limited access to RNA-based NGS or other restrictions (costs, long distance/ time transportation, DNA based laboratory sample collections for retrospective analysis). Further studies should help to evaluate the relevance of the technology in the clinical practice.Keywords: Mutation analysis, Kinase domain mutant, Chronic myeloid leukemia, BCR::ABL